Plant growth is dictated by both internal and external factors. The internal mechanisms originate in the genetic makeup of the plant and influence the extent and timing of its growth. These internal mechanisms are regulated by signals of various types transmitted within the plant cells, between the cells, or all around the plant itself. The external factors are directly related to the immediate environment surrounding the plant. These external influences affect plant growth and include factors such as water, nutrients, temperature, light, fertilizers, pesticides, bio-stimulants etc. The external environment can place constraints on the extent to which internal mechanisms can permit the plant to grow and develop, with two of the most important factors being related to the availability of water and nutrient supplies in the soil.
Cell expansion is directly related to water supply, and thus any deficit results in a smaller plant. Mineral nutrients are needed for the biochemical processes of the plant. When nutrients are in insufficient supply, growth will be less vigorous, or in extreme cases, it will cease altogether. The nutrients necessary for plant growth include the primary macronutrients Nitrogen (N), Phosphorous (P), and Potassium (K); the secondary macronutrients Calcium (Ca), Sulphur (S), and Magnesium (Mg); and the micronutrients or trace minerals Boron (B), Chlorine (Cl), Manganese (Mn), Iron (Fe), Zinc (Zn), Copper (Cu), Molybdenum (Mo), and Selenium (Se). Optimal temperatures are also necessary for plant growth. The required temperature range will depend on the species, but most plants grow slowly at low temperatures, i.e., 0° C. to 10° C., and some tropical plants are damaged or even killed at low but above-freezing temperatures. Light is also important in the control of plant growth, in that it drives the process of photosynthesis.
Plant bio-stimulants are components other than fertilizers that affect plant growth and/or metabolism upon foliar application or when added to soil. Plant bio-stimulants generally fall within one of these categories i.e. hormone-containing products, plant extract based products, micronutrients based products, amino acid-containing products and humic acid-containing products but may not be strictly restricted to these categories alone. Plant bio-stimulants are used to treat crops in a commercial setting in view of their ability to increase growth rates, increase stress tolerance, increase photosynthetic rate and increase disease tolerance. Plant bio-stimulants are generally believed to operate by up-regulating or down-regulating plant hormones.
Despite the commercially availability of numerous fertilizers and plant bio-stimulants, there continues to be a demand for improved products capable of serving a variety of needs. Therefore new products and methods for improving plant growth responses and development are needed.
Adhatoda zeylanica (commonly known as Adhatoda vasica, Malabar Nut, Adulsa, Adhatoda, Vasa, Vasaka, Vasica) belonging to family Acanthaceae is a small, evergreen shrub found mainly in regions of India and throughout the world, with a multitude of uses in traditional Ayurveda. Apart from Ayurveda, this herb has been used for treatment of various diseases and disorders in Unani also. Vasica is most well-known for its effectiveness in treating respiratory conditions. The leaves of Vasica are known to show stimulant effect on the respiratory system. Vasica shows an antispasmodic and expectorant effect, and has been used for centuries with much success to treat asthma, chronic bronchitis, and other respiratory conditions. The phytochemical studies of the various parts of Adhatoda zeylanica revealed the presence of alkaloids, phytosterols, polyphenolics and glycosides as a major class of compounds. Its principal constituents are quinazoline alkaloids with Vasicine as its main alkaloid.
In agriculture, extracts of Adhatoda zeylanica bark, leaves, and flower have also been used to eradicate living red spider mites and eggs from leaves, where this plant has also shown certain efficacy. However, there were no reports concerning the use of Adhatoda zeylanica as bio-stimulant, which can be defined as substance that is neither a plant nutrient nor a pesticide, but has a positive impact on plant health. In other words, a bio-stimulant is an organic material that, when applied in small quantities, enhances plant growth and development such that the response cannot be attributed to application of traditional plant nutrients.
Moringa oleifera (also called Horseradish tree or Drumstick tree) is a plant originally found in the Himalayan regions of India. The plant belongs to the family of Moringaceae. It is a shrub and small deciduous tree of 2.5 m to 10 m in height. When matured, the fruit becomes brown and has 10-50 seeds inside (Vlahof et al., 2002). This plant is popularly grown in Africa, the Middle East, southeastern Asia, the Pacific Islands, the Caribbean Islands and southern America and is now widely planted in Taiwan and China. In India, the leaf and fruit of M. oleifera were originally used as vegetables with the root acting as a substitute for horseradish in cooking. This plant was also reported to contain various amino acids, fatty acids, vitamins, and nutrients (Nesamani, 1999). The constituents of the M. oleifera tree such as the leaf, flower, fruit, and bark have been anecdotally used as herbal medicines in treatments for inflammation, paralysis, and hypertension. Moreover, many reports indicate that M. oleifera has highly potent anti-inflammatory (Ezeamuzle et al., 1996), hepatoprotective (Pari and Kumar, 2002), antihypertensive (Faizi et al., 1995) and antitumor (Murakami et al., 1998) properties in humans. Also, its seed has strong coagulative and anti-microbial properties (Eilert et al., 1981). The seed oil has physical and chemical properties equivalent to that of olive oil and contains a large quantity of tocopherols (Tsaknis et al., 1999). The leaf extracts in rats were found to regulate thyroid status and cholesterol levels (Tahiliani et al., 2000; Ghasi, 2000). Therefore it can be developed as a useful agent to treat high cholesterol. The screening of various parts of M. oleifera revealed the presence of various phytochemicals among which quercetin is the main flavonoid.
The use of Moringa oleifera extract as a biostimulant in enhancing the growth, biochemical and hormonal contents in rocket plants (Eruca vesicaria subsp. sativa) has been well documented (Int. J. Plant Physiol. Biochem. Vol. 5(3), pp. 42-49, September 2013).
Algae, seaweed, and seaweed derived products have been widely used in agriculture as an enhancer due to the presence of a number of plant growth stimulating compounds in them. Galactans, carrageenans and agars present in cell wall of red seaweeds are known to aid seed germination, elongate root and retain moisture in the soil. Furthermore, seaweed extracts are also a rich source of macronutrients (N, P, K, Ca, S, Mg) and micronutrients (Fe, Mn, Co, Cu) which are present in metalloprotein active sites.
Various methods of extraction of Vasicine from Adhatoda zeylanica extract are known in the art. According to a prior art process Vasicine can be isolated from the leaves of Adhatoda zeylanica by extracting the leaves of the plant with 95% alcohol, treating the concentrated alcoholic extract with aqueous 2% H2SO4, basifying the aqueous acidic solution with ammonia and extracting with chloroform, concentrating the chloroform gave an extract which was again dissolved in aqueous 2% H2SO4 and repeating the process of basification with ammonia, followed by extraction with chloroform (C. K. Atal. “Chemistry and Pharmacology of Vasicine—A new oxytoxic and abortifacient.” Raj. Bandhu Ind. Co., New Delhi, 1980). The drawback of the process include use of strong mineral acid like H2SO4 for extraction which results in considerable degradation of Vasicine, which is further aggravated by repeating the process of same mineral acid treatment twice.
In another prior art, Vasicine was isolated from Adhatoda zeylanica leaves as follows (D. R. Mehta, J. S. Naravane and R. M. Desai. Vasicinone. A Bronchodilator Principle from Adhatoda Vasica Nees (N. O. Acanthaceae) J. Org. chem. 28, 445-448, 1963). The leaves were refluxed with 90% alcohol and after evaporation of the solvent, the alcohol extract thus obtained was extracted with hot distilled water and the aqueous extract was filtered. The filtrate was extracted with chloroform to remove the coloring matter and then made alkaline with 5% caustic soda, and again extracted with chloroform. The combined chloroform extracts were extracted with 5% hydrochloric acid, and then acidic solution was made alkaline with ammonia and again extracted with chloroform. After repeating the process twice the final chloroform extract was concentrated to give a crude total alkaloid from which Vasicine was isolated as Vasicine hydrochloride (yield: 2 g). The first drawback of the above process includes the extraction of the alcohol extract with hot water, with further limitations like (a) Vasicine could not be quantitatively extracted from its aqueous solution and (b) hot water extraction will convert Vasicine into its auto oxidation product vasicinone. Second drawback of the process is the use of 5% mineral acid like hydrochloric acid for its extraction and that also, twice. The use of strong mineral acid degrades Vasicine considerably and thus results in a lower yield of Vasicine.
U.S. Pat. No. 6,676,976 B2 discloses an improved process for the production of Vasicine from the Adhatoda vasica said process comprising the steps of extracting the dried and pulverized leaves with an alcoholic extract at an ambient temperature, concentrating the alcoholic extract to obtain a concentrated extract, treating and stiffing extract with an aqueous organic acid for 2-24 hours, extracting the acid solution with an organic solvent, separating the organic layer and aqueous acidic layer, basifying the aqueous acidic solution with a base, extracting the basified solution with an organic solvent, separating the organic layer, drying and filtering, evaporating the organic layer to obtain an amorphous residue, and treating the amorphous residue with an organic solvent or mixture of organic solvents to obtain Vasicine. Even this process suffers from the drawbacks of using corrosive acids and low yields.
In view of the above discussion, there is a need for a process that extracts Vasicine from Adhatoda zeylanica plant in an effective manner. There is also a need to explore the use of Adhatoda zeylanica extract containing Vasicine as a bio-stimulant for plant growth promotion and identify its suitable formulation for application in agriculture.